Determination of serine proteases

ABSTRACT

Serine proteases in a substance such as blood are determined by contacting the substance with a novel chromogenic or fluorgenic substrate for serine proteinases and spectrophotometrically measuring the quantity of a chromophore or fluorescent compound released from the substrate by serine proteases in the substance. The novel substrate has the amino acid sequence-ILe-A-Gly-Arg-wherein A is Asp or Glu substituted in the carboxylic group by esterification or amidation. The novel substrate increases sensitivity and accuracy of the determination.

This is a division of application Ser. No. 852,006, filed Nov. 16, 1977,now U.S. Pat. No. 4,207,232.

This invention relates to new chromogenic substrates for serineproteases. The new substrates are suitable for the determination offactor Xa (E.C. 3, 4, 21.6) or for the study of reactions which cause Xato be formed, inhibited or consumed or even for the determination ofsuch factors that influence or participate in such reactions.

Factor X is a key substance in the series of reactions leading to thecoagulation of blood. The activation of factor X brings about theformation of the proteolytic enzyme, factor Xa, which is directlyresponsible for the transference of prothrombin to thrombin. Thetransformation of prothrombin to thrombin by factor Xa involves thecleavage of two peptide bonds in the prothrombin molecule. These twocleavage sites are preceded by exactly the same amino acid sequence:-Ile-Glu-Gly-Arg-.

A simple method for the determination or coagulation factor Xa is veryvaluable for diagnostic purposes. The hitherto very best reagents forthe determination of factor Xa consist of chromogenic peptide substrateswith an amino acid sequence, -Ile-Glu-Gly-Arg-, corresponding to thesequence preceding the cleavage sites of the natural substrateprothrombin.

The substrate Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222), which isthe best substrate in this series, has an amino acid sequence identicalto the above discussed part of the natural substrate. This substrate hasalready found application in clinical assays of e.g. antifactor Xa andHeparin (A. N. Teien, M Lie and U Abildgaard, Thrombosis Research 8,413, 1976). The methods are based on the following reaction: ##STR1##The released p-nitroanilide (pNA) has a light absorption maximumdifferent from that of the substrate, and the enzymatic reaction caneasily be followed by measuring the increase in absorption at 405 nm,which is proportional to the amount of active factor Xa.

A large number of synthetic substrates for factor Xa has been describedby L Aurell, G Claeson, G Karlsson and P Friberger in Peptides 1976, p191, Proc. from the XIVth European Peptide Symposium, Weipon, Belgium,1976. The amino acids in the natural sequence were varied. They were inturn replaced by other similar amino acids, but in no case a bettersubstrate than S-2222 was obtained. Even very small changes in thenatural sequence, such as replacement of Ile by Leu, give substrateswith much lower sensitivity.

The new chromogenic substrates, according to the invention, arerepresented by the following general formula:

    R.sub.1 -Ile-A-Gly-Arg-NH-R.sub.2

or salts thereof, wherein R₁ is acyl, preferably acetyl or benzoyl; R₂is p-nitrophenyl, β-naphthyl or 4-methoxy-β-naphthyl, i.e. aromaticgroups which give a chromophore or fluorescent compound, R-NH₂, at theenzymatic hydrolysis; A is Asp or Glu, substituted in the carboxylicgroup preferably by esterification or amidation. Suitable esters containa short alkyl-, hydroxyalkyl-, substituted aminoalkyl- or cycloalkylgroup. The esters employed contain 2-6 carbon atoms. Suitable amidescontain mono- or disubstituted short alkyl-, hydroxyalkyl- orsubstituted aminoalkyl group, or a heterocyclic group in which the amidonitrogen forms part of a piperidine, morpheline or piperazine ring. Theamides employed contain 1-6 carbon atoms.

The natural γ-carboxy group of Glu, which at biological pH has anegative charge and is hydrophilic, has according to the invention, beenreplaced by a considerably larger group, which is neutral andlipophilic. Since previous changes of the natural sequence.-Ile-Glu-Gly-Arg-, have only given inferior substrates (L Aurell, GClaeson, G Karlsson and P Friberger in Peptides 1976, p 191, Proc. fromthe XIVth European Peptide Symposium, Weipon, Belgium, 1976), it is verysurprising that the comparatively big changes made in the invention haveresulted in substrates with fundamentally better properties (seeTable 1) than the previously best substrate, S-2222. The drasticlowering of the Michaelis constant (Km) is the most striking, and forpratical work, the most important improvement achieved with the newsubstrates. Km is defined as the substrate concentration required forthe attainment of half of the maximal velocity (Vmax). The previoussubstrates and also the substrates according to the invention havelimited solubilities, and for that reason the substrate concentrationshould be below 1 mM when working in buffer solution and blood plasmaunder practical conditions. With S-2222, which has Km=0.83 mM, one canonly make full use of half of Vmax, whereas with the substrates in theinvention, which have a 2 to 5 times lower Km, one can make much betteruse of the maximum velocity. Thus, obvious advantages are obtained whenmeasuring factor Xa activity with the new substrates. Their sensitivityis several hundred percent higher than that of S-2222, which makespossible a much higher accuracy, a fundamentally lower sensitivity limitand a strongly decreased volume of the blood sample.

Assays of enzymes with the help of chromogenic substrates are verysuitable for use in autoanalyzers, and the higher sensitivity of thesenew substrates brings about a shorter reaction time in the apparatus andwith that also the possibility to analyze more samples per time-unit.

The new substrates according to the invention may be produced fromchromogenic or fluorogenic substrates containing the amino acidsequences -Ile-Glu-Gly-Arg- and -Ile-Asp-Gly-Arg-, respectively, byesterification or amidation of the free γ- or β-carboxylic group withmethods which are well known and commonly used in peptide chemistry.

The following examples illustrate the invention.

In the thin layer chromatographic analysis of eluates and products,glass plates with silica gel F₂₅₄ (Merck) were used as the absorptionmedium. The solvent system used is chloroform, methanol, acetic acid andwater in the volume ratios 34:4:9:2.

After the thin layer chromatographing, the plates were studied first inUV-light (254 nm) and subsequently by using thechlorine/toluidine-reaction (G Pataki, Dunnschichtchromatografie in derAminosaure- und Peptidchemie, Walter de Gruyter & Co Berlin, p 125,1966) as a development method.

The meanings of the abbreviations used below are as follows:

Amino acids:

These abbreviations refer to amino acid rates. The free amino acid orpeptide is indicated by means of H-- at the amino group and --OH at thecarboxyl group. The amino group is always indicated to the left, thecarboxyl group to the right.

Unless otherwise stated, all amino acids used have the L-configuration.

Arg=Arginine

Asp=Aspartic acid

Glu=Glutamic acid

Gly=Glycine

Ile=Isoleucine

Leu=Leucine

Further abbreviations:

Ac=Acetyl

AcOH=Acetic acid

Bz=Benzoyl

DCCI=Dicyclohexylcarbodiimide

DMF=Dimethylformamide

Et₃ N=Triethylamine

HOBT=α-Hydroxybenzotriazol

HOSu=N-Hydroxysuccinimide

MeOH=Methanol

OEt=Ethyloxy

OMe=Methyloxy

OpNp=p-Nitrophenoxy

OisoPr=iso-Propyloxy

pNA=p-Nitroanilide

QAE=Quaternary amino-ethylsepharose (Pharmacia Fine Chemicals)

SOCl₂ =Thionylchloride

EXAMPLE 1 Bz-Ile-Glu(OMe)-Gly-Arg-pNA.HCl (M.w. 748.2)

Under moisturefree conditions, at 0° C., 30 μl of distilled SOCl₂ isadded to 0.5 ml of absolute methanol. After the first lively reactionthe solution is left for about 15 min at room temperature, and 75 mg(0.10 mmole) of Bz-Ile-Glu-Gly-Arg-pNA.HCl (S-2222) is added. Thesolution is stirred for 5 hours and then evaporated. The oil obtained isdissolved in a small amount of methanol and purified by gelchromatography on a column containing Sephadex LH-20 (Pharmacia FineChemicals) in methanol with methanol as eluating means. The pure methylester, obtained after evaporation of the methanol, is dissolved in waterand lyophilized.

Yield: 30 mg (40%)

Homogenous according to TLC, R_(f) =0.33

[α]_(D) ²⁰ -40.3° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 2 Bz-Ile-Glu(OEt)-Gly-Arg-pNA.HCl (M.w. 762.2)

Under moisturefree conditions, at -10° C., 60 μml or distilled SOCl₂ isadded to 1.0 ml of absolute ethanol. After 30 min at room temperature150 mg of S-2222 is added. When the reaction is finished after about 15hours (according to TLC), the solution is evaporated. The oil obtainedis dissolved in a small amount of 30% HOAc in H₂ O and is purified bychromatography on a column containing Sephadex G 15 (Pharmacia FineChemicals) in 10% HOAc in H₂ O. The pure ethyl ester from the eluate islyophilized.

Yield: 70 mg (46%)

Homogenous according to TLC, R_(f) =0.40

[α]_(D) ²⁰ -37.7° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 3 Bz-Ile-Glu(OisoPr)-Gly-Arg-pNA.HCl (M.w. 776.3)

The synthesis is performed according to the method of Example 2 butabsolute isopropanol is used instead of ethanol. The reaction iscompleted after 16 hours. Chromatography and lyophilization is performedaccording to these procedures in Example 2.

Yield: 90 mg (58%)

Homogenous according to TLC, R_(f) =0.44

[α]_(D) ²⁰ -36.4° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 4 Bz-Ile-Glu(O-cyclohexyl)-Gly-Arg-pNA.HCl (M.w. 833.3)

60 μl SOCl₂ is added to 1.0 ml of dry cyclohexanol, and after 1 hour atroom temperature 200 mg S-2222 is added. When the reaction is finishedafter about 12 hours the solution is evaporated and chromatographedaccording to these procedures in Examples 2 and 3, whereupon the productis lyophilized.

Yield: 170 mg (75%)

Homogenous according to TLC, R_(f) =0.50

[α]_(D) ²⁰ -38.6° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 5 Bz-Ile-Glu(O-CH₂ CH₂ N(CH₃)₂)-Gly-Arg-pNA.HCl (M.w. 859.8)

75 mg (0.10 mmole) of S-2222 and 100 mg (0.8 mmole) ofdimethylaminoethanol hydrochloride are dissolved in 1.0 ml of drydistilled DMF, whereupon 10 μl of pyridin, 10 mg of HOBT (0.074 mmole)and finally 25 mg (0.12 mmole) of DCCI are added. The dicyclohexylureaformed is filtered after about 24 hours, and the DMF solution evaporatedunder reduced pressure. The remaining oil is dissolved in a small amountof 95% MeOH--5% H₂ O and purified on a column containing QAE-25 ionexchanger in its chloride form. The same solvent mixture is used aseluant. By this procedure the hydrochloride of the dimethylaminoethylester is obtained free from other peptides, but contains some minorimpurities, e.g. dimethylaminoethanol hydrochloride. The fractioncontaining the dimethylaminoethyl ester is evaporated and purified bychromatography on a column containing Sephadex G15 (Pharmacia FineChemicals) in 10% HOAc/H₂ O. The solution of the pure ester islyophilized. Yield: 60 mg (58%)

Homogenous according to TLC, R_(f) =0.50

[α]_(D) ²⁰ -35.0° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 6 ##STR2##

240 mg (0.33 mmole) of S-2222 and 45 mg (0.39 mmole) of HOSu aredissolved in 1 ml of dry distilled DMF. The solution is cooled to -5° C.and 120 mg (0.58 mmole) of DCCI is added. The temperature is allowed torise to room temperature and after 4 hours the solution is again cooleddown to 0° C. and the precipitated dicyclohexylurea is filtered andwashed. The DMF solution (about 2 ml) is cooled to 0° C. and 0.1 ml ofpure isopropylamine is added. After about 70 hours at room temperaturethe solution is evaporated under reduced pressure, mixed with 5 ml ofwater and again evaporated. The product is dissolved in about 4 ml of50% HOAc/H₂ O and is purified by chromatography on a column containingSephadex G15 (Pharmacia Fine Chemicals) in 33% HOAc/H₂ O. The samesolvent mixture is used as eluant. The faction containing the pureisopropylamide is evaporated and is ion-exchanged on a column containingquaternary aminoethylsepharose, QAE25 (Pharmacia Fine Chemicals) in itschloride form in 95% MeOH 5% H₂ O. The eluate is evaporated, dissolvedin water and lyophilized.

Yield: 120 mg (47%)

Homogenous according to TLC, R_(f) =0.39.

[α]_(D) ²⁵ -30.6° (c 0.5, MeOH)

EXAMPLE 7 ##STR3##

The synthesis is performed according to the method of Example 6 butpiperidine is used instead of isopropylamine.

Yield: 105 mg (40%)

Homogenous according to TLC, R_(f) =0.50

[α]_(D) ²⁵ -34° (c 0.5, MeOH)

EXAMPLE 8 ##STR4##

The synthesis is performed according to the method of Example 6 butdiethanolamine is used instead of isopropylamine.

Yield: 120 mg (45%)

Homogenous according to TLC, R_(f) =0.25

[α]_(D) ²⁵ -31° (c 0.5, MeOH)

EXAMPLE 9 Bz-Ile-Asp(OisoPr)-Gly-Arg-pNA.HCl (M.w. 762.3)

30 μl of distilled SOCl₂ is added to 0.5 ml of dry isopropanol, andafter 30 min at room temperature 73 mg (0.10 mmole) ofBz-Ile-ASP-Gly-Arg-pNA.HCl (M.w. 720.2) is added. When the reaction isfinished after about 18 hours the solution is evaporated,chromatographed and lyophilized according to these procedures inExamples 2, 3 and 4.

Yield: 32 mg (42%)

Homogenous according to TLC, R_(f) =0.46

[α]_(D) ²³ -22.7° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 10 Bz-Ile-Asp(OEt)-Gly-Arg-pNA.HCl (M.w. 748.2)

The synthesis is performed according to the method of Example 9 butethanol is used instead of isopropanol.

Yield: 28 mg (37%)

Homogenous according to TLC, R_(f) =0.40

[α]_(D) ²³ -23.5° (c 0.5, 50% HOAc/H₂ O)

EXAMPLE 11 ##STR5##

The synthesis is performed according to the method of Example 6 butBz-Ile-Asp-Gly-Arg-pNA.HCl is used as starting material instead ofS-2222.

Yield: 100 mg (40%)

Homogenous according to TLC, R_(f) =0.40

[α]_(D) ²⁵ -20.1° (c 0.5, MeOH)

EXAMPLE 12 ##STR6## The synthesis is performed according to the methodof Example 11, but morpholine is used instead of isopropylamine.

Yield: 95 mg (35%)

Homogenous according to TLC, R_(f) =0.46

[α]_(D) ²⁵ -24.2° (c 0.5, MeOH)

Determination of Km and Vmax

Km and Vmax are obtained by means of the Lineweaver-Burk equation:##EQU1## Enzyme and substrate are mixed in a buffer solution and therate of hydrolysis is measured spectrophotometrically. The substrateconcentration (S_(o)) is varied while the enyzme concentration is keptconstant. The reciprocal velocity l/v_(o) is then plotted against thereciprocal substrate concentration l/S_(o), and Km and Vmax areestimated (Table 1) from the obtained Lineweaver-Burk diagram.

    ______________________________________                                        Reagents                                                                      Buffer  Tris (tris(hydroxymethyl)aminomethane)                                        0.05 mole/l, pH = 8.3, I = 0.25 (NaCl)                                Enzyme  Diagen (6.4 Denson U) (Diagnostic Reagents)                                   . 1 ampule is dissolved in 2 ml of water.                             Substrate                                                                             The substrate is dissolved in water to 2 mmole/l.                     Method  Buffer, +37° C.2400-1850 μl                                         Enzyme, +20° C. 50 μl  2500 μl                                   Substrate, +37° C.50-600 μl                                         Mix and read change of absorbance (Δ OD/min) at                         405 nm and at 37° C.                                           Relative                                                                              Buffer, +37°  C.2200 μl                                     activity                                                                              Enzyme, +20° C.50 μl                                                Substrate, +37° C.250 μl                                            Mix and read change of absorbance (Δ OD/min) at                         405 nm and at 37° C.                                           ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                        Km, Vmax and Relative activities                                                        Km · 10.sup.4                                                                    Vmax · 10.sup.8                                                                  Relative                                    Substrate mol/l       mol/min · U                                                                      activity                                    ______________________________________                                        S-2222    8.3         8.6         100                                         Ex. 1     2.6         8.1         230                                         Ex. 2     2.9         7.5         200                                         Ex. 3     2.1         7.1         220                                         Ex. 6     5.6         20.0        340                                         Ex. 7     1.7         8.6         300                                         ______________________________________                                    

What we claim is:
 1. A method for diagnostic determination of serineproteases which comprises contacting a substance in which saiddetermination of serine poteases is desired with a chromogenic orfluorogenic enzyme substrate containing the sequence -Ile-A-Gly-Arg-R₂where A is Asp or Glu, substituted in the carboxylic group byesterification or amidation, wherein the esters contain a short chainalkyl or hydroxyalkyl or alkyl-substituted aminoalkyl or cycloalkylgroup containing a total of 2-6 carbon atoms, and the amides containmono or di-substituted short chain alkyl or hydroxyalkyl oralkyl-substituted aminoalkyl group containing a total of 1-6 carbonatoms or a heterocyclic group in which the amido nitrogen forms part ofa piperidine, a morpholine or a piperazine ring, and R₂ is a group whichgives a chromophore or fluorescent compound upon enzymatic hydrolysis,and then spectrophotometrically measuring the quantity of chromophore orfluorescent compound released from said substrate by enzymatichydrolysis of the substrate by serine proteases in said substance. 2.The method of claim 1 wherein said serine proteases is factor Xa.
 3. Themethod of claim 1 wherein said substrate has the formula R₁-Ile-A-Gly-Arg-NH-R₂ wherein R₁ is acyl.
 4. The method of claim 3wherein R₁ is acetyl.
 5. The method of claim 3 wherein R₁ is benzoyl. 6.The method of claim 3 wherein R₂ is p-nitrophenyl or β-naphthyl or 4methoxy-β-naphthyl or salt thereof.
 7. The method of claim 1 wherein Ais Asp or Glu substituted with a methoxy group.
 8. The method of claim 1wherein A is Asp or Glu substituted with an ethoxy group.
 9. The methodof claim 1 wherein A is Asp or Glu substituted with an isopropoxy group.10. The method of claim 1 wherein A is Asp or Glu substituted with acyclohexoxy group.
 11. The method of claim 1 wherein A is Asp or Glusubstituted with a dimethylaminoethoxy group.
 12. The method of claim 1wherein A is Asp or Glu substituted with CONHCH(CH₃)₂.
 13. The method ofclaim 1 wherein A is Asp or Glu substituted with ##STR7##
 14. The methodof claim 1 wherein A is Asp or Glu substituted with CON(CH₂ CH₂ OH)₂.15. The method of any of claims 7-14 wherein said substrate has theformula R₁ -Ile-A-Gly-Arg-NH-R₂ wherein R₁ is benzoyl and R₂ isp-nitrophenyl.